Tobacco ringspot virus (TRSV) is listed as a quarantine (Q) pest in the European Union (EU) because it can cause major losses in blueberry (Vaccinium corymbosum), grapevine (Vitis vinifera) and soybean (Glycine max) productions (EFSA PLH Panel et al., 2019). Although Q-pests are supposed to be absent in the EU, TRSV has been reported worldwide, including several Member States (MSs), such as Czech Republic (Kundu et al., 2016), Italy (Bellardi & Marani, 1985; Sorrentino et al., 2012), Lithuania (Samuitienė & Navalinskienė, 2001), the Netherlands (Asjes, 1974), Poland (Kaminska, 1985), and the former MS United Kingdom (UK) (DEFRA, 2018; Stone, 1980). TRSV is a member of the genus Nepovirus, subgroup A, consisting of two single-stranded RNA segments that both contain a single open reading frame (ORF) encoding for a polyprotein. It has a wide host range, including many ornamentals. The virus is naturally spread by Xiphinema americanum sensu lato (s.l.) nematodes, which occur around the world, but from this species complex only X. rivesi has been reported in a few EU MSs (EFSA PLH Panel et al., 2018; EPPO, 2022b). Apart from transmission by EU populations of X. rivesi under experimental conditions (Sirca et al., 2007), natural spread of TRSV has not been reported in the EU, despite occasional findings of TRSV. The first report of TRSV in ornamentals in the Netherlands (Asjes, 1974), together with incidental findings of TRSV between 1997 and 2006, prompted the Netherlands Institute for Vectors, Invasive plants and Plant health (NIVIP) and the Netherlands Inspection Service for Horticulture (Naktuinbouw) to set up nationwide survey programs for TRSV and its vector species. Here we report the results of these surveys and other incidental TRSV findings since 1997. Additionally, the results of a phylogenetic analysis of complete genome sequences of available TRSV isolates from these findings and from NCBI GenBank are discussed in a phytosanitary context.
In 2006, a TRSV survey program was set up in ornamentals (Table 1), based on previous findings, reports in literature and notifications from other countries. Standard samples were taken regardless of symptoms and consisted of leaf material from five plants per lot. For Ajuga reptans, Hemerocallis, Iris germanica, and Phlox subulata (2017-2020), samples consisted of leaf material from 200 plants per lot, tested in subsamples from 5 plants. For the bulbous crops, both leaf and root material were sampled and tested.
Detection and identification of TRSV was done by bioassay, DAS-ELISA, real-time RT-PCR, and Illumina sequencing. Bioassays were performed by inoculating plant sap from leaf material on Chenopodium quinoa, Nicotiana benthamiana and Nicotiana occidentalis P1(EPPO, 2022a). DAS-ELISA was either performed on leaf material of the original host or on symptomatic leaf material from inoculated plants using the TRSV antiserum from Prime Diagnostics (Wageningen, The Netherlands) (EPPO, 2015).
A real-time RT-PCR was designed, based on in silico analyses of GenBank accessions (see Annex 1): primers TRSV-F1 (TGT TAA TGT CCA TTT TTG GTA GAA ART G), TRSV-F2 (TGT TGA TGT CTA TTT TTG GTA GAA AGT G), TRSV-R (TAA GTG CAC ACC CCG ACG G), and TRSV-P (FAM-TCC CAA GTT TTT GAA GTT CGG GGT GG-BHQ1). After grinding leaf material in GH+ buffer (Botermans et al., 2013), RNA was isolated, using the Sbeadex® maxi plant kit (LGC genomics) on a KingFisher KF96 system following the manufacturer’s instructions. Reaction mixtures consisted of 6.25 µl UltraPlex™ 1-Step ToughMix® (4x) (Quanta Biosciences, Plain City, Ohio, USA), 0.15 µM of each forward primer, 0.3 µM of the reverse primer, 0.1 µM of the probe, 2 µl RNA extract, an internal RNA isolation control (nad5) (Botermans et al., 2013), i.e., 0.02 µM of each primer and 0.1 µM of the probe, and RNAse-free water added up to a final volume of 25 µl. Real-time RT-PCR was performed in 96-well plates on a Bio-Rad CFX96™ real-time PCR Detection System (Bio-Rad Laboratories Hercules, California, USA): 10 min 50 °C, 3 min 95 °C, 40 cycles 10 s 95 °C and 1 min 60 °C. Results were analyzed using BioRad CFX Maestro 2.0 software (Bio-Rad Laboratories). The test was validated according to EPPO standard PM 7/98 (5) Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity (EPPO, 2021). Analytical sensitivity: relative dilution 10-5 for natural infected material of Pelargonium and Vaccinium. Analytical specificity: all TRSV isolates included in this study were detected, and in naturally infected plant material no cross reactions were observed with other nepoviruses, i.e., Arabis mosaic virus, beet ringspot virus, blueberry leaf mottle virus, cherry leaf roll virus, grapevine fanleaf virus, potato black ringspot virus, raspberry ringspot virus, and tomato ringspot virus. Selectivity: no matrix effects observed, except for hosts with viscous plant sap (e.g., Phlox subulata); matrix effects could be prevented by diluting plant sap homogenates 2-5 times in GH+ buffer before RNA extraction. Repeatability and reproducibility were100%. The real-time RT-PCR assay was designed in 2019, based on 40 TRSV sequences (NCBI GenBank and Q-bank). In 2023, 109 TRSV sequences, available from NCBI GenBank, were aligned with the primers and probe: 95% of the sequences had no or one mismatch(es) with the primers or probe (Figure S1) Three sequences with more than one mismatch were tested using synthetic DNA-fragments (Invitrogen) and were all detected, although with less sensitivity for the most deviating sequence (Table S1). Illumina sequencing was performed as described by Schoen et al. (2023).
Table 1. List of ornamental species included in surveys for TRSV in the Netherlands from 2005-2020, including number of samples per year, 2005-2020
Host
|
Yeara
|
|
05
|
06
|
07
|
08
|
09
|
10
|
11
|
12
|
13
|
14
|
15
|
16
|
17
|
18
|
19
|
20
|
Ajuga reptans
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
30
(8)
|
|
Allium (ornamental)
|
|
|
|
|
|
|
|
|
32
|
|
|
|
|
|
|
|
Anemone
|
|
|
|
|
15
|
|
|
|
|
|
|
|
|
|
|
|
Astilbe
|
|
|
|
|
|
|
|
3
|
3
|
|
6
|
|
|
|
|
|
Bacopa
|
|
35
(2)
|
40
|
|
|
|
|
3
|
6
|
|
5
|
|
|
|
|
|
Celosia
|
|
|
|
35
(7)
|
|
|
4
|
12
|
7
|
|
|
|
|
|
|
|
Gladiolus
|
|
|
|
61
|
92
|
53
|
30
|
|
|
|
|
|
|
|
|
|
Gypsophila
|
|
|
|
|
|
|
|
|
6
|
|
|
|
|
|
|
|
Hemerocallis
|
|
39
(1)
|
13
(9)
|
25
|
8
|
8
|
|
|
|
|
|
|
|
30 (7)
|
|
|
Hosta
|
|
14
|
|
35
|
20
|
14
|
|
|
|
|
|
|
|
|
|
|
Hydrangea
|
|
|
|
|
|
|
3
|
6
|
6
|
2
|
5
|
|
|
|
|
|
Iris chrysographes
|
|
1
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Iris ensata
|
|
150
(25)
|
|
|
|
|
|
|
|
|
|
|
7
|
|
|
|
Iris germanica
|
|
|
|
|
|
|
|
|
|
|
|
|
9
(1)
|
30
(14)
|
|
|
Iris hollandica
|
|
1
|
|
|
|
|
|
|
|
|
28
|
26
|
32
|
|
|
|
Iris louisiana
|
|
15
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Iris pallida
|
|
1
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Iris pseudacorus
|
|
10
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Iris siberica
|
|
10 (1)
|
|
|
|
|
|
|
|
|
|
|
10
|
|
|
|
Iris spuria
|
|
1
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Iris versicolor
|
|
4
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Iris virginica
|
|
2
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Lilium
|
|
|
|
60
|
54
|
45
|
30
|
30
|
30
|
30
|
30
|
30
|
30
|
30
|
30
|
30
|
Lobelia
|
|
|
42
|
39
|
|
|
|
|
|
|
|
|
|
|
|
|
Narcissus
|
120
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Pelargonium
|
70
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Pentas
|
|
|
|
|
|
|
|
6
|
6
|
|
|
|
|
|
|
|
Phlox subulata
|
|
|
|
46
|
58
(1)
|
50
(5)
|
|
5
|
|
|
7
|
|
|
|
30
|
|
Portulaca
|
|
19
(1)
|
36
(5)
|
|
|
|
|
|
3
|
3
|
|
4
|
|
|
|
|
Tulipa
|
|
|
|
|
|
|
45
|
30
|
30
|
|
|
|
30
|
30
|
30
|
30
|
a Years in which TRSV was identified are in bold, indicating number of samples and, between brackets, the number of positive lots
Samples were sequenced on a NextSeq 500 or NovaSeq 6000 platform as 150 paired-end reads. During the surveys in 2017-2019 samples from Ajuga, Hemerocallis, and I. germanica were analyzed by Illumina sequencing. Isolates from outbreaks before 2017 were sequenced in 2021 when collection material was still available. Sequences of each genome segment were aligned with available (nearly) complete RNA1 and RNA2 sequences of TRSV from NCBI GenBank using MAFFT (Katoh et al., 2002) in Geneious Prime 2021.1.1 (Biomatters, New Zealand). External gaps and missing sequences were recoded as question marks. Phylogenetic analyses were performed with Bayesian inference (BI) using MrBayes v. 3.2.6 (Huelsenbeck & Ronquist, 2001). To determine the optimal model to construct the phylogenetic trees, for each genome segment a model test was performed in CLC genomic workbench (Qiagen, Denmark), using default settings. Subsequent analysis was performed with a random starting tree and four Monte Carlo Markov chains for 1.1x106 generations under the GTR model with G distribution. Trees were sampled every 200 generations. The first 5x104 generations were discarded as burn-in, and the remaining trees combined to generate a 50% majority rule consensus tree that represented posterior probabilities.
The possible presence of X. americanum s.l. was determined by collecting soil samples from randomly selected orchards, fruit tree nurseries and flower-bulb fields, as well as from fields of growers where TRSV was identified in Hemerocallis, Iris ensata, and I.germanica (Table 2). Soil sampling was performed with an auger of 2 cm diameter and 40 cm depth. From each field an area of 1/3 ha was selected and a composite sample of 60 cores was taken in a systematic grid pattern, resulting in c. 7 litre soil (EPPO, 2009). The soil samples were transported to the laboratory and kept at 4°C until processing. From each thoroughly mixed composite soil sample a subsample of 200 ml was taken for nematode extraction by Oostenbrink elutriation with settings and sieves for extraction of larger nematodes (EPPO, 2013). The extracted suspension was morphologically analyzed on the presence of Xiphinema spp..
Table 2. List of Xiphinema spp. Surveys, from 2006-2021
Year
|
Sampling locations
|
Number of samples
|
2006-2007
|
Fields from growers with TRSV infected plants of: Iris, Hemerocallis
|
56
|
2013-2016
|
Orchards of: Malus, Pyrus, Prunus avium, Prunus domestica, Vitis
|
230
|
2017-2020
|
Nurseries of: Malus, Prunus, Pyrus
|
93
|
2018
|
Fields from growers with TRSV infected plants of: Hemerocallis, Iris
|
18
|
2018-2021
|
Flower bulb fields of: Tulipa, Lilium
|
90
|
Table 3 gives an overview of the findings of TRSV in ornamentals in the Netherlands from 1997 until 2021. The findings in Lobelia (1997) and Bacopa, and Portulaca (2000) followed shortly after these plants were imported from Israel. The survey and findings in Hemerocallis and Iris spp. In 2006-2007 were related to a notification from the United States of America. Further surveys resulted in the identification of additional TRSV isolates, except for the isolate from Celosia, which was analyzed because of a TRSV notification. In all cases, TRSV-infected plants were asymptomatic, except for a few Hemerocallis plants (2006) which appeared to be co-infected by a novel putative Luteovirus (results not shown). In the surveys in the Netherlands, TRSV was not detected in ornamental Allium, Anemone, Astilbe, Gladiolus, Gypsophila, Hosta, Hydrangea, Iris chrysographes, I. hollandica, I. louisiana, I. palida, I. pseudacorus, I. spuria, I. versicolor, I. virginica, Lilium, Narcissus, Pelargonium, Pentas, and Tulipa. In samples from imported plant material, TRSV was identified in Ajuga reptans, Coleus, Pelargonium zonale, and Salvia rosmarinus (Table 4). Regarding the surveys for Xiphinema spp., the absence of X. americanum s.l. in the Netherlands was confirmed. Xiphinema species were present in 26 of the 481 soil samples, 25 samples from fruit production orchards and one sample from a Pyrus nursery. In seventeen of these samples the nematodes could be determined as X. diversicaudatum. In the other nine samples only Xiphinema juveniles were found, which were not X. americanum s.l.. Therefore, based on the absence of X. americanum s.l., vegetative propagation is considered the main mode of spread of TRSV in the Netherlands.
Table 3. TRSV findings in the Netherlands between 1997-2021
|
|
Detected in number of
|
Detection/ identification method
|
|
Year
|
Host species
|
Lots
|
Cultivars
|
ELISA
|
Real time RT-PCR
|
HTS
|
NCBI genbank accession numbersa
|
1997
|
Lobelia
|
5
|
- b
|
X
|
X
|
X
|
ON229950/1
|
2000
|
Bacopa
|
1
|
1
|
X
|
X
|
X
|
ON229952/3
|
2000
|
Portulaca
|
35
|
- b
|
X
|
X
|
X
|
ON229954/5
|
2006
|
Bacopa
|
2
|
- b
|
X
|
|
|
-
|
2006-2007
|
Portulaca
|
33
|
- b
|
X
|
X
|
|
-
|
2006-2007
|
Hemerocallis
|
22
|
4
|
X
|
X
|
X
|
ON229942/3
|
2006-2007
|
Iris ensata
|
25
|
21
|
X
|
X
|
X
|
ON229934/5
|
2006-2007
|
Iris siberica
|
1
|
1
|
X
|
|
|
-
|
2008
|
Celosia
|
7
|
- b
|
X
|
X
|
X
|
ON229932/3
|
2009-2010
|
Phlox subulata
|
6
|
2
|
X
|
X
|
X
|
ON229936/7
|
2017-2018
|
Iris germanica
|
15
|
4
|
X
|
X
|
X
|
ON229968/9
|
2018
|
Hemerocallis
|
7
|
1
|
X
|
X
|
X
|
ON229960/1
|
2019
|
Ajuga reptans
|
8
|
2
|
X
|
X
|
X
|
ON229956/7
|
a NCBI accession numbers of only one isolate per outbreak are included; accession numbers are written as RNA1/RNA2, RNA2 has the subsequent NCBI accession number. b Cultivar(s) of original host unknown
Table 4. TRSV findings from imported plant material
Year
|
Host species
|
Country of origin
|
ELISA
|
Real time RT-PCR
|
HTS
|
NCBI genbank accession numbera
|
2018
|
Ajuga reptansb
|
Guatemala
|
X
|
X
|
X
|
ON229946/7
|
2019
|
Pelargonium zonale
|
United Kingdom
|
X
|
X
|
X
|
ON229948/9
|
2019
|
Ajuga reptansb
|
Germany
|
X
|
X
|
X
|
ON229944/5
|
2019
|
Coleus
|
United states of Americac
|
X
|
|
X
|
ON230014/5
|
2020
|
Salvia rosmarinus
|
Israel
|
X
|
X
|
X
|
ON230016/7
|
a NCBI accession numbers included as RNA1/RNA2, RNA2 has the subsequent NCBI accession number. b Different cultivar than the Dutch isolate. c Plant was intercepted in Germany
Phylogenetic analyses of all generated and available GenBank sequences show that isolates cluster different for their RNA1 and 2 sequences (Figure 1), for example in the RNA1 tree MT563080 from Glycine max clusters with ON229972 from Hemerocallis and ON230016 from S. rosmarinus, while for RNA2 this sequence clusters with the different sequences from Cucumis melo. Although sequence clustering is different for RNA1 and 2, there are several groups where sequences cluster together in both trees, indicating that these isolates might have a common origin. For both RNAs A. reptans sequences cluster together with the sequences from Bacopa, Celosia, Lobelia, and Portulaca. Although Celosia was imported from Denmark, while A. reptans, Bacopa, Lobelia, and Portulaca were imported from Israel, the similarity in TRSV sequences suggest that they all might have a similar origin. Only the A. reptans sequence from Guatemala clusters outside this group, suggesting a different origin of the isolate. Furthermore, as could be expected sequences from C. melo from the same field in Texas cluster together (Tabara et al., 2021), as well as the sequences of Hemerocallis from 2017-2018. In contrast, the sequences from Hemerocallis from 2006-2007 do not cluster with those from 2017-2018, and sequences from I. germanica cluster in different clades which is more apparent for RNA1 than for RNA2. The clusters from I. germanica are not cultivar specific, i.e., sequences from the same cultivars group in different clusters and most clusters contain sequences of at least two cultivars. Overall, the similarity in sequences within the same host and year of finding, irrespectively of location, substantiates that TRSV was spread by vegetative propagation. Additionally, the fact that genotypes differ between different plant species indicates that TRSV had been able to enter the Netherlands on multiple occasions.
The observed difference in clustering of RNA1 and 2 of TRSV is in line with Hily et al. (2021), who recently showed that the phylogenetic relationship between the Nepovirus subgroups was different when looking at either RNA1 or RNA2. While the division between subgroup A, B and C was clear when comparing RNA1 sequences, this was less clear for RNA2. RNA2 sequences of subgroup A, of which TRSV is a member, and C appeared scattered over different clades. Our results further substantiate this different clustering behavior of RNA1 and 2 and show the importance of using both RNA’s to determine phylogenetic relationships.
The results of the TRSV surveys revealed that the virus occurs asymptomatic in various ornamental species.. The phylogenetic analyses showed that TRSV likely entered the Netherlands on multiple occasions and had further spread by vegetative propagation. The previously mentioned reports of TRSV in ornamentals from several EU MSs, suggest that this situation is likely similar in other EU MSs. This is substantiated by the identification of TRSV in Celosia from Denmark and Ajuga and Coleus from Germany in this study. The approach of asymptomatic surveys might explain why findings in ornamentals are more frequent in the Netherlands compared to other EU MSs. Therefore, systematic surveys in ornamentals regardless of symptoms are needed to determine the pest status in the EU, but since these have not been carried out by most other MSs, the pest status is undetermined. Furthermore, the likely unnoticed spread by vegetative propagation in the EU underlines the importance of using virus-free material for vegetative propagation. Additionally, the TRSV reports in ornamentals raise the question whether the EU Q status is still applicable for TRSV. To qualify for a Q status, the pest needs to be absent in the EU, or if present, to have a restricted distribution, and have an unacceptable economic, social or environmental impact after introduction (Regulation (EU) 2016/2031). This study indicates that TRSV is probably more widespread in the EU than currently reported, at least in ornamental plants, and therefore does not meet the criteria of a Q-pest. Moreover, the risks associated with the unnoticed presence in ornamentals seems limited, although an argument could be made that this is an unacceptable risk because the virus could establish by X. rivesi from these sources. Current legislation does not offer protection against the possibility of transmission by X. rivesi from ornamentals to the crops at risk, i.e., blueberry, grapevine and soybean. This implies that preventing the establishment of TRSV, requires large scale surveys in ornamentals and subsequent eradication measures in all MSs. The question is whether it is desirable for EU phytosanitary agencies to put large efforts on this virus. The number of Q-pest outbreaks has been increasing over the past years (European commission, 2022), while resources are limited. Therefore, another option which could be considered, is to regulate TRSV as a Regulated non-Quarantine pest (RNQP) for blueberry, grapevine, and soybean. Moreover, in comparison to the current Q-status, an RNQP status will provide better guarantees of the absence of TRSV as it requires testing of plants for planting of those crops at risk. In view of these considerations, the evaluation of the regulatory status of TRSV is highly recommended.